Vol.3 No.2 2010

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Research paper : Development of an accurate and cost-effective quantitative detection method for specific gene sequences (N. Noda)−166−Synthesiology - English edition Vol.3 No.2 (2010) has the same strand length as the target gene, and also is amplified by the same primer and the fluorescent alternately binding probe (AB-Probe). One terminal of the AB-Probe is labeled with the green fluorescent dye (BODIPY FL) where the fluorescence is quenched with the nearby guanine base, and the other end is labeled with the red fluorescent dye (TAMRA) that is quenched by the guanine base. The sequence of the AB-Probe is designed to be complementary to the common sequence of the target gene and the internal standard gene, and it hybridizes with the same affinity to both genes. In the internal standard gene, the three exterior bases on the green fluorescent dye that hybridize with the AB-Probe are replaced with the guanine base (the bases in the target gene are those other than guanine). Therefore, the AB-Probe binds competitively to the amplified product derived from the target gene and that from the internal standard gene with the same affinity, and green fluorescence is emitted when it binds with the target gene, but does not emit fluorescence when it binds with the internal standard gene since the fluorescent dye is quenched by the guanine base. The green fluorescence becomes stronger as there are more target genes than the internal standard genes, while the green fluorescence becomes weaker as there are less target genes than the internal standard genes. The amount of the target gene can be calculated since the amount of the internal standard gene is known. TAMRA, the red fluorescent dye, is quenched in the same manner when the AB-Probe is bonded either to the target gene or the internal standard gene. The presence of amplification can be checked since the degree of quenching of TAMRA changes according to the amount of amplified product derived from the target gene and the internal standard gene.The ABC-PCR can be considered as a method where the electrophoresis step that was mandatory in the competitive PCR method is replaced with the fluorescent probe using the quenching phenomenon by guanine. Since it is a competitive method, it not only allows accurate quantification in the presence of the PCR inhibitors, but is also an endpoint quantification method where the degree of quenching can be measured after the completion of PCR. Therefore, the expensive device needed in the RT-PCR is not necessary, and the target gene can be quantified with an inexpensive thermal cycler and a fluorescence measurement device.3 Results of the development3.1 Universal QProbe PCR methodWe conducted an experiment to verify the principle of the universal QProbe PCR by using the -actin, albumin, and -globin genes as target genes. The most important point is the stability of the joint DNA and the fluorescent probe. It is desirable that the hybridization between the joint DNA and the fluorescent probe does not dissociate but remain stable during the PCR reaction. Therefore, we used a synthetic oligonucleotide where the nucleic acid of the fluorescent probe was replaced by locked nucleic acid (LNA). The LNA is an analog of the nucleic acid that has two cyclic structures in the molecule, and it is known that the oligonucleotides including LNA show dramatic heat stability against the complementary DNA and RNA[11]. A fluorescent probe labeled with BODIPY FL composed of LNA of 13 base length was synthesized, and the Tm of the complementary sequence of this fluorescent probe and the joint DNA was calculated using the Exiqon Tm prediction tool (http://lna-tm.com). The result was 102 ºC. Since the highest temperature encountered in PCR was 95 ºC at heat denaturation, it was thought that the complex of the fluorescent probe and joint DNA would be prevented from dissociation during the PCR cycle.Using the designed fluorescent probe and the joint DNA, the quantification of the target gene was conducted using the universal QProbe PCR. The fluorescent quenching rate was calculated from the fluorescence value at denaturation step (state where the probe and target genes are dissociated) and the fluorescence value during annealing step (state where the probe and target genes are bonded). Figure 4 shows 00101020202530303540404550-551510Number of cyclesQuenching rate (%)Initial template108 copies107106105104103102N01020304050Number of cyclesAmount of initial templates (copies)1.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+09y = 8E +10e-0.5341x R2 = 0.9967Fig. 4 Relationship between the number of cycles and quenching rate in the universal QProbe methodThis shows the quenching rate when the -actin gene is amplified from 10 to 108 copies. The calculation of quenching rate was done according to Reference [8].Fig. 5 Standard curve in the universal QProbe methodThis shows the relationship between the number of cycles required for the reaction product to reach a certain amount and the amount of initial templates, calculated from the relationship between the number of cycles and quenching rate of Fig. 4.

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